Angiogenesis
Angiogenesis is the process in which new blood vessels grow into an area which lacks a sufficient blood supply. Angiogenesis commences with the erosion of the basement membrane surrounding endothelial cells and pericytes forming capillary blood vessels. Erosion of the basement membrane is triggered by enzymes released by endothelial cells and leukocytes. The endothelial cells then migrate through the eroded basement membrane when induced by angiogenic stimulants. The migrating cells form a “sprout” off the parent blood vessel. The migrating endothelial cells proliferate, and the sprouts merge to form capillary loops, thus forming a new blood vessel.
Angiogenesis can occur under certain normal conditions in mammals such as in wound healing, in fetal and embryonic development, and in the formation of the corpus luteum, endometrium and placenta. Angiogenesis also occurs in certain disease states such as in tumor formation and expansion, or in the retina of patients with certain ocular disorders. Angiogenesis can also occur in a rheumatoid joint, hastening joint destruction by allowing an influx of leukocytes with subsequent release of inflammatory mediators.
The evidence for the role of angiogenesis in tumor growth was extensively reviewed by O'Reilly and Folkman in U.S. Pat. No. 5,639,725, the entire disclosure of which is incorporated herein by reference. It is now generally accepted that the growth of tumors is critically dependent upon this process. Primary or metastatic tumor foci are unable to achieve a size of more than approximately 2 mm in the absence of neovascularization. Serial evaluation of transgenic mice predisposed to develop neoplasms has demonstrated that neovascularization of premalignant lesions precedes their evolution into tumors (Folkman et al., Nature 339:58-61, 1989), and that inhibition of angiogenesis delays the growth of such lesions, as well as their assumption of a malignant phenotype (Hanahan et al., Cell 86:353-364, 1996). In humans, several studies have demonstrated that increased density of microvessels within a tumor is associated with a poor clinical outcome (Weidner et al., J Natl Cancer Inst 84:1875-1887, 1992).
An emerging paradigm is that proteolytic fragments of plasma or extracellular matrix proteins regulate angiogenesis. To date, several polypeptides with such activities have been identified. These include angiostatin, which contains kringles 1-4 plasminogen (O'Reilly et al., Cell 79:315-328, 1994), endostatin, a 20 kD C-terminal fragment of collagen XVIII (O'Reilly et al., Cell 88:277-285, 1997), PEX, the hemopexin domain of matrix metalloprotease 2 (Brooks et al., Cell 92:391-400, 1998), the C-terminal 16 kD fragment of prolactin (Clapp et al., Endocrinol 133:1292-1299, 1993) and a 29 kD fragment of fibronectin (Homandberg et al., Am J Pathol 120:327-332, 1985). In addition, both intact thrombospondin 1 as well as peptides derived from its procollagen domain and properdin-like type-1 repeats express potent anti-angiogenic activity (Good et al., Proc Nat Acad Sci USA 87:6624-6628, 1990); Tolsma et al., J Cell Biol 122:497-511, 1993. In preclinical models, several of these fragments inhibited tumor growth, and some induced tumor regression and dormancy (Boehm et al., Nature 390:404-407, 1997).
High Molecular Weight Kininogen
High molecular weight kininogen (HK) is a 120 kD glycoprotein containing heavy and light chains, comprised of domains 1 through 3, and 5and 6, respectively (Kaplan et al., Blood 70:1-15, 1987). The heavy and light chains are linked by domain 4, which contains bradykinin, a nonapeptide which mediates several events including NO-dependent vasodilation (Weimer et al., J Pharm Exp Therapeutics 262:729-733, 1992). HK (also referred to as “single chain high molecular weight kininogen”) binds with high affinity to endothelial cells, where it is cleaved to two-chain high molecular weight kininogen (HKa) by plasma kallikrein. Bradykinin is released from HK through cleavage mediated by plasma kallikrein (Kaplan et al., Blood 70:1-15, 1987). This event occurs on the surface of endothelial cells following the activation of prekallikrein to kallikrein by an endothelial cell protease (Motta et al., Blood 91:515-528, 1998). Cleavage of HK to form HKa and release bradykinin occurs between Lys(362) and Arg(363). HKa contains a 62 kD heavy chain and a 56 kD light chain linked by a disulfide bond.
Conversion of HK to HKa is accompanied by a dramatic structural rearrangement, which has been demonstrated using rotary shadowing electron microscopy (Weisel et al., J. Biol Chem 269:10100-10106, 1994). HKa, but not HK, has been shown to inhibit the adhesion of endothelial and other cell types to vitronectin (Asakura, J. Cell Biol 116:465-476, 1992). HKa, but not HK, also binds tightly to artificial anionic surfaces.
HK domain 3 consists of HK amino acids Gly(235)-Met(357). HK domain 3 has the following amino acid sequence:                Gly-Lys-Asp-Phe-Val-Gln-Pro-Pro-Thr-Lys-Ile-Cys-Val-Gly-Cys-Pro-Arg-Asp-Ile-Pro-Thr-Asn-Ser-Pro-Glu-Leu-Glu-Glu-Thr-Leu-Thr-His-Thr-Ile-Thr-Lys-Leu-Asn-Ala-Glu-Asn-Asn-Ala-Thr-Phe-Tyr-Phe-Lys-Ile-Asp-Asn-Val-Lys-Lys-Ala-Arg-Val-Gln-Val-Val-Ala-Gly-Lys-Lys-Tyr-Phe-Ile-Asp-Phe-Val-Ala-Arg-Glu-Thr-Thr-Cys-Ser-Lys-Glu-Ser-Asn-Glu-Glu-Leu-Thr-Glu-Ser-Cys-Glu-Thr-Lys-Lys-Leu-Gly-Gln-Ser-Leu-Asp-Cys-Asn-Ala-Glu-Val-Tyr-Val-Val-Pro-Trp-Glu-Lys-Lys-Ile-Tyr-Pro-Thr-Val-Asn-Cys-Gln-Pro-Leu-Gly-Met (SEQ ID NO:18).        
HK binds to endothelial cells, platelets and neutrophils in the intravascular compartment. A specific cell attachment site has been identified on HK domain 3 by an antibody-directed strategy utilizing an antibody HKH15, selected for its ability to block HK binding to cells (Herwald et al., J. Biol Chem 270:14634-14642 (1995). A series of HK domain 3 synthetic peptides was examined for ability to inhibit biotin-HK from binding to human umbilical vein endothelial cells. As a result, the cell binding site was localized to a domain 3 segment containing HK amino acids Leu(331)-Met(357). Other weakly inhibiting peptides include Lys(224)-Pro(254), Asn(276)-Ile(301) and Leu(331)-Met(357). However, the effect on endothelial cell proliferation was not studied.